Bacterial sortases were originally identified as enzymes that covalently attach proteins to the bacterial cell wall. For example, Staphylococcus aureus sortase A recognizes a set of diverse substrates via a sortase recognition motif (e.g., LPXTG) and cleaves the peptide bond between threonine and glycine, thereby releasing the residues C-terminal to the threonine and yielding an amide linkage with the N terminus of a pentaglycine nucleophile, which is provided in vivo by a cell wall precursor.
The transpeptidation reaction catalyzed by sortases has emerged as a versatile method for site-specific modification of proteins and has been applied to a variety of in vitro reactions. The method has proved versatile in part because the enzyme tolerates a wide variety of substrates in proximity of the cleavage site and in nucleophiles. In many sortase-based protein modification methods a protein to be modified is engineered to contain a sortase recognition motif (e.g., LPXTG) at or near its C-terminus. When incubated with sortase and a synthetic peptide containing one or more N-terminal glycine residues, such artificial sortase substrates undergo a transacylation reaction resulting in the exchange of residues C-terminal to the threonine residue with the synthetic peptide, resulting in the protein C-terminus being ligated to the N-terminus of the synthetic peptide. In some cases, a protein to be modified is engineered to contain one or more N-terminal glycine residues near its N-terminus. When incubated with sortase and a synthetic peptide containing a sortase recognition motif and a sortase, the transacylation reaction results in the exchange of residues C-terminal to the threonine residue in the synthetic peptide with the modified protein, resulting in the synthetic peptide being ligated to the N-terminus of the protein. The synthetic peptides used in either approach may be fused or conjugated to any of a number of different moieties. When the synthetic peptide and protein are conjugated via sortase-mediated transacylation, such moieties become attached to the protein.
The sortase-catalyzed reaction has been used for, among other things, ligating proteins and/or peptides to one another in vitro, conjugating a protein or peptide to a solid support or polymer, and linking a label to a protein or peptide.